Post on 22-Jun-2020
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Lecture 8: Molecular Markers and mapping
Read 69-78 (ie. 2.6-2.7)
Fig. 2.22-2.27
Table 2.4
DNA polymorphism: the basis of molecular markers
Methods of detection and application (diagnosis,finger-printing)
Molecular mapping
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Four classes of DNA polymorphisms
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Single nucleotide polymorphism (SNP)
• Single base-pair substitutions• Arise by mutagenic chemicals or mistakes in
replication• Biallelic – only two alleles• Ratio of alleles ranges from 1:100 to 50:50• 2001 – over 5 million human SNPs identified• Most occur at anonymous loci• Mutation rate of 1 X 10-9 per locus per generation• Very few are thus new mutation in the species• Useful as DNA markers
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Microsatellites• 1 every 30,000 bp• Repeated units 2 – 5 bp
in length• Mutate by replication
error• Mutation rate of 10-3
per locus per gamete• Useful as highly
polymorphic DNAmarkers
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Minisatellites
• Repeating units20-100 bp long
• Total length of0.5 – 20 kb
• 1 per 100,000bp, or about30,000 in wholegenome
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Deletions, duplications, and insertions
• Expand or contract the length ofnonrepetitive DNA
• Small deletions and duplications arise byunequal crossing over
• Small insertions can also be caused bytransposable elements
• Much less common than otherpolymorphisms
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Ler
Col
HindIII HindIII HindIII
HindIII HindIII
2 kb 5.7 kb
Ler Col Het1kb
ladder
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2
1
Probe:
RFLP: Restriction Fragment Length Polymorphism
HindIII digestionElectrophoresisSouthern blottingHybridization with the probeautoradiography
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• SNP detection usingsouthern blots– Restriction fragment length
polymorphisms (RFLPs)are size changes infragments due to the loss orgain of a restriction site
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Example of RFLP
Restriction digestionGEEthedium bromide stain
BlottingHybridizationX-ray radiography
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CAPS (Cleaved Amplified Polymorphic Sequences) ie. RFLP detection by PCR
Ler
Col
HindIII HindIII HindIII
HindIII HindIII
2 kb 5.7 kb
Ler Col Het1kb
ladder
5
2
1
PCRHindIII digestionGE
L C M
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CAPS
• Must have sequence oneither side ofpolymorphism– Amplify fragment– Expose to restriction
enzyme– Gel electrophoresis
• e.g., sickle-cellgenotyping with a PCRbased protocol
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Randomly amplified polymorphic DNA(RAPD)
PCR is done with asingle short primer that
hybridizes to manyplaces in a genome.
Occasionally, 2primers hybridize to
complementary strandsnear each other
generating a PCRproduct. Different
organisms of aspecies have slightly
different DNA sequenceswhich yield mostly thesame PCR products,
but a few PCR productsare gained or lost.
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SNP detection by ASO
• Very short probes (<21 bp) specific which hybridize to one allele or other• Such probes are allele-specific oligonucleotides (ASOs)
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Large-scale ASO analysis with microarrays
Each column contains an ASO differing only at thenucleotide position under analysis
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Primer extension to detect SNPs
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Detecting microsatellite markers
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L C 1 2 3
Microsatellite allele detection by PCR
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Huntington’sdisease is anexample of amicrosatellitetriplet repeatin a codingregion
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Minisatellite detection and DNAfingerprinting
• 1985 – Alec Jeffreys made two key findings– Each minisatellite locus is highly polymorphic– Most minisatellites occur at multiple sites around the
genome– DNA fingerprint – pattern of simultaneous genotypes at
a group of unlinked loci– Use restriction enzymes and southern blots to detect
length differences at minisatellite loci– Most useful minisatellites have 10 – 20 sites around
genome and can be analyzed on one gel
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• Minisatelliteanalysis
Fig. 11.14
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• DNA fingerprints can identifyindividuals and determineparentage
• E.g., DNA fingerprintsconfirmed Dolly the sheepwas cloned from an adultudder cell
• Donor udder (U), cell culturefrom udder (C), Dolly’s bloodcell DNA (D), and controlsheep 1-12
Dolly the sheep is a clone of adult udder cell